Xylocaine

Twenty adult, male, Wistar rats, weighing between 250 and 300g, were used in this study. The animals were kept in a proper room, with light-dark cycles of 12 hours. Housed in proper cages, they had free access to food and water. All the procedures were carried out according to the recommendations from the Ethics Committee in Research of the São Paulo Hospital – Federal University of São Paulo (UNIFESP)
These rats were anesthetized by intraperitoneal injection of 2% Xylazine (0.5ml/kg) and 10% ketamine (0.9ml/kg). After that, the hair from the right side retroauricular region was removed. They were then placed in lateral decubitus and received an injection of xylocaine (it was repeated if necessary) in this region, and then we started the surgical procedure, which resulted in right side peripheral facial paralysis in these animals. The procedure was carried out by two surgeons, through the use of surgical microscopes DF-Vasconcellos M90, with a 200mm lens and 16x magnification. Surgical results were photographed with a 3.2 megapixels Sony Cybershot camera and were standardized.
All the animals were observed as to spontaneous and stimulated facial movements. This assessment was carried out by the same observer, and occurred before the surgery (to rule out facial movement alterations present prior to surgery that could compromise the later assessment), and in alternate days in the postoperative time, during 30 days. The parameters observed were: eye closure, blinking reflex, vibrissae movement and positioning. To do that, each rat was individually evaluated in a cardboard box (37×21×13cm) painted black inside (to better visualize the vibrissae), and as stimulation we used air inflation (a 20 ml syringe) on the animal face to trigger the blinking reflex, and hand clapping (three to four times) to cause vibrissae movement. The left side served as the control side (blinking reflex present with complete eye closure; normal and correctly positioned vibrissae movement). Through this observation we created a scale to assess facial movement in these animals.

EGD was performed by 14 experienced endoscopists. EGD was performed as a screening method during a health evaluation, for follow-up of gastritis and/or gastric tumor, for the examination for abdominal symptoms, to investigate an abnormality of photofluorography, to examine abnormal serum pepsinogen levels, or due to a positive finding of H. pylori antibody. The pharynx of the patients was topically anesthetized with a gargle of lidocaine hydrochloride 2% viscous solution (Xylocaine^® Viscous 2%; AstraZeneca Inc., Cambridge, UK) before the EGD.^{(7 )} The endoscopists were allowed to use their clinical judgement to decide the amount and type of sedative and analgesic medication and the antagonist—midazolam (0–10 mg), pethidine (0–70 mg), flumazenil (0–0.5 mg) and naloxone (0–0.4 mg)—to be used. Following the EGD, the patients were transferred to the recovery room. All adverse events including nausea and vomiting were evaluated by the recovery room nurse. Patients were requested to return 10 to 14 days later for the explanation of their EGD results and were also interviewed regarding any additional adverse events.

Upon completion of baseline testing, FLEX, INT and INFLEX rats were randomly assigned to the sham or lesioned group. Rats were anesthetized with isoflurane (2% at 1.5 l/min) and placed in a stereotaxic frame (Kopf Instruments®, Tujunga, CA, USA). Vitamin A was applied on the eyes to prevent corneal dryness and 1 mL of saline solution was subcutaneous injected to prevent dehydration. Pre-surgical analgesia was performed with ketoprofen (10 mg/kg, i.p.) and local analgesia with 2% xylocaine gel after disinfection with vetedine®. Two bilateral injections were performed in the SNc (in millimeters from bregma and dura and according to the Paxinos and Watson rat brain atlas, AP: −5.1, ML: +/−2.2, DV: −8 and AP: −5.6, ML: +/−2; DV: −8) with 1 µL of AAV2 expressing human A53T α-synuclein (CMVie/SynP-synA53T-WPRE, 5.2 10¹³ gcp/mL) or AAV2 expressing GFP (green fluorescent protein, CMVie/SynP-GFPdegron-WPRE, 3.7 10¹³ gcp/mL) at 0.2 µL/min as previously described^{19 ,35 (link)}. Post-surgical analgesia was performed with ketoprofen (10 mg/kg/day, i.p.) the day after surgery. Akinesia was estimated twice a day during four consecutive days using the stepping test as previously described^{36 (link)} at baseline, 4 and 9 weeks after surgery, and following chronic PPX treatment.